1. Elucidating the role of MBP1 in the pathogenesis of Candida albicans Gregory J. Fischer Julie Anderson Daniel Herman Department of Biology University of Wisconsin–Eau Claire Abstract Pathogenesis Plasmid Construction and Isolation MBP1 Protein Expression Experimental Results Conclusions Experimental Design  Candida albicans MBP1 is functionally equivalent to Saccharomyces cerevisiae MBP1 and is able to rescue the lethal phenotype of a mbp1/swi4 double mutant, thus playing a role in the cell cycle.  Suggests that C. albicans MBP1 activates genes in the G1/S transition that are orthologous to those targeted by S. cerevisiae MBP1.  Is evidence that the MBP1 protein could potentially serve as a drug target for the next generation of antifungal agents.  Cases of fungal infections, such as oropharyngeal candidiasis (OPC) continue to be problematic, especially in neonatal intensive care units (Dotis, et al., 2010).  Recent studies of ICU and cancer patients consistently find C. albicans to be the predominant yeast species isolated (Figure 2) (Shoham, et al., 2009 & Lalla, et al., 2010).  There is evidence that MBP1 plays a role in the filamentous growth of C. albicans and is similar in sequence to the S. cerevisiae gene (Herman, et al., in progress). Genotype of Yeast Strains Used in Study YJJ1068 (Betz, et al., 2002) Mat α leu2∆1 his3∆200 ura3-52 mbp1∆::KNMX4 [pLEU-MBP1] YJJ1000 (Betz, et al., 2002) Mat a leu2 ∆1 his3∆200 ura3-52 swi4∆::URA3 JA2011 Mat a/α leu - /leu - ura + /ura - mbp1 + /mbp1∆ swi4+/swi4∆ [pLEU-MBP1] GJF2011 leu2∆1 his3∆200 ura3-52 swi4∆::URA3 mbp1 ∆::KNMX4 [ pLEU-MBP1] The yeast species Candida albicans is the most commonly-isolated yeast in human disease. Systemic infections of C. albicans have emerged as important causes of mortality in premature infants and in the immunocompromised, with the number of cases on the rise. To infect host tissue, the usual unicellular form of C. albicans switches into an invasive, multicellular filamentous form. This morphogenesis or conversion to the filamentous state has been shown to contribute significantly to C. albicans’ pathogenicity. We have been investigating the role of the MBP1 gene in this process. The MBP1 homolog in the nonpathogenic yeast Saccharomyces cerevisiae has been well studied and plays a role in the G1-S transition within the cell cycle. To further our understanding of the function of MBP1 in C. albicans, we are expressing C. albicans’ MBP1 within a S. cerevisiae MBP1 knockout strain to assess whether the MBP1 protein carries out similar functions in both organisms. Experimental results will help elucidate the role MBP1 plays in morphogenesis which could lead to novel therapies for these types of infections. Molecular Genetics of Yeast MBP1 Leu2 P-Gal pESC-Leu This faculty/student research collaboration was made possible through Differential Tuition and a grant from the Office of Research and Sponsored Programs. Acquiring Double Mutant YJJ1068YJJ1000 JA2011 Sporulation (JA2011)  Separate spores by micro-dissection.  Identify genotype via replica plating. Role of MBP1 in Cell Cycle Figure 1: Fungal infections, such as OPC, continue to be problematic, especially within NICUs, with antifungal resistant strains emerging. Figure 2: Prevalence of Candida species in fungal infections for ICU and cancer patients. Recent studies continue to identify C. albicans as the predominant yeast species isolated in fungal infections. Meiotic Product Characteristics GenotypePhenotype MBP1+/Swi4+Leu+/Ura - /G418 S MBP1+/swi4ΔLeu+/Ura+/G418 S mbp1Δ/Swi4+Leu+/Ura - /G418 R mbp1Δ/swi4Δ (LETHAL)Leu+/Ura+/G418 R Figure 5: The use of homologous recombination for the creation of a vector construct. C. albicans MBP1 was placed downstream of a Gal promoter, allowing for conditional expression of MBP1 in the presence of galactose. MBP1 Swi4 WT YSMG, Duke Univ. Image Courtesy C. Halde Shoham, et al., 2009 Lalla, et al., 2010 References Alberts B., D. Gray, J. Lewis, et al., (1994). Molecular Biology of the Cell 3 rd Edition. New York: Garland Science. Betz, J.L., M. Chang, T.M. Washburn. S.E. Porter, C.L. Mueller, & J.A. Jaehning (2002). Phenotypic analysis of Paf1/RNA polymerase II mutations reveals connections to cell cycle regulation, protein synthesis, and lipid and nucleic acid metabolism. Mol Genet Genomics, 268:272-285. Dotis, J. & E. Roilides (2010). Candidemia in the pediatric intensive care unit: What’s different from candidemia in adults? Current Fungal Infection Reports, 5(1): 49-55. Jorgenson, P., J.L. Nishikawa, B. Breitkreutz, & M. Tyers (2002). Systemic Identification of pathways that couple cell growth and division in yeast. Science, 297:395-400. Lalla, R.V. M.C. Latortue, C.H. Hong, et al., (2010). A systemic review of oral fungal infection in patients receiving cancer therapy. Support Care Cancer, 18:985-992. Shoham, S. & S. Marwaha (2009). Invasive fungal infections in the ICU. Journal of Intensive Care Medicine, 25(2):78-92. Figure 4: Following transformation, plasmid DNA was isolated and cut to determine whether the MBP1 insert was present. The results indicate that homologous recombination was successful in DNA of colonies 2 and 5. mbp1Δ/swi4Δ Leu-MBP1 Vector Only Master PlateGalactoseGlucose mbp1Δ/swi4Δ [pLEU-MBP1-1] Vector Only Figure 8: Replica plating revealed that a double mutant yeast strain grew on galactose media but not glucose. Yeast with functional S. cerevisiae MBP1 transformed with vector only was used as a positive control. Immunoblot of the double mutant strain confirmed C. albicans MBP1 protein expression. Therefore, C. albicans MBP1 is able to functionally replace the MBP1 ortholog in S. cerevisiae. Immunoblot Figure 3: A) Schematic of the activation or inhibition of SBF and MBF by various proteins within S. cerevisiae. During growth phases, in which the size of the cell increases, transcription factor complexes like SBF and MPF are inhibited so that cell division does not occur. Once conditions merit division, SBF and MBF are directly involved with the activation of genes responsible for DNA replication and bud emergence. B) MBP1 and Swi4 interact with the adapter protein Swi6 to form the MBF and SBF complexes respectively. Jorgenson, et al., 2002 SBF/MBF CLN1/2 Bud emergence (CLA4, BEM1, BEM2, BEM4) DNA replication SPB duplication Swi6 Swi4 SBF Complex Swi6 MBP1 MBF Complex A) B) Alberts, et al., 1994 A) YJJ1000 mbp1 + /swi4Δ Gene Disruptions Ura3Swi4 KNMX4MBP1 YJJ1068 swi4 + /mbp1Δ B) Figure 6: A) Immunoblot using chemiluminescence to detect C. albicans MBP1 protein under the control of a GAL promoter. Primary and secondary antibodies were used at concentrations of 1:1000 and 1:5000, respectively. These results confirmed that C. albicans MBP1 expression is induced by galactose. B) Schematic of a typical immunoblot using chemiluminescence. A myc-epitope was added to MBP1 in the expression system and an anti-myc antibody was used to detect the protein. Leu-MBP1UraUra-MBP1 UraLeu Leu-MBP1 Ura-MBP1 Leu GlucoseGalactose MBP1 ~94kDa A) Luminol & H 2 O 2 Mouse anti-myc Donkey anti-mouse HRP Enzyme Light myc MBP1 myc-fusion protein 93.9 kDa B) MBP1 insert ~2.5kb mbp1Δ/swi4Δ [pLEU-MBP1-2] Mating n 2n n Figure 7: A) In the budding yeast lifecycle, haploid yeast cells of opposite mating types undergo conjugation to produce a diploid yeast strain. Mitosis of this diploid strain continues until meiosis is triggered by lack of essential nutrients. Sporulation of the diploid cell results in four haploid meiotic products known as spores. The genotypes of these spores can be determined by separating and growing them on selective media. A mbp1/swi4 double mutant was isolated using this technique in order to assess the activity of C. albicans MBP1. B) Gene disruptions of MBP1 via Geneticin (G418) resistance cassette (KNMX4) in YJJ1000 strain and Swi4 disruption using a URA3 cassette in YJJ1068 strain. Growth of these strains on media containing Geneticin (G418) or lacking uracil indicates that genomic MBP1 and Swi4 are disrupted.